Human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), enters cells by binding its envelope glycoprotein (Env) to receptor molecules and fusing its membrane with the cell membrane. Understanding how receptor molecules mediate HIV entry is critical for elucidating the mechanisms of HIV infection, and design of new antiretroviral treatments and vaccines. We have previously found that the Env forms a complex with the primary receptor CD4 and coreceptor molecules (CCR5 and CXCR4). During the last year we have been further characterizing the interactions in the HIV-1 Env-CD4-coreceptor complex leading to entry. We found that the cell surface concentration of Env-CD4-CCR5 complexes correlates with fusion and that increasing surface concentration of CCR5 results in an increased concentration of CD4-CCR5 complexes in proportion to fusion. We developed a new model system based on immunopurified CCR5, CD4 and Env. By using this system our collaborators R. Blumenthal and A. Dimitrov showed that purified CCR5-CD4 complexes can induce conformational changes in the Env resulting in cell membrane permeabilization likely due to exposure of the fusion peptide. These results not only provide basis for the identification and characterization of a new fusion intermediate but also suggest that the conformations of CCR5 and CD4 in our model system are close to their native conformation. We demonstrated the native conformation of CCR5 by another independent experiment which showed that RANTES was able to bind CCR5 associated through an antibody to protein G sepharose beads. By using these purified complexes we immunized mice transgenic in human CD4 and CCR5 and found antibodies against gp120 but no neutralization activity. Currently we are preparing complexes which also will include gp41 with a major goal of development of an HIV vaccine able to elicit cross-reactive neutralizing antibodies. Our ability now to produce these complexes in relatively large amounts would allow us to immunize rabbits and monkeys. We also studied the mechanism of action of 20 small molecule HIV inhibitors and several peptide inhibitors hypothesizing that they may disrupt the Env-CD4-coreceptor complexes. The results suggest that some of them do but others do not. Interestingly some of the inhbitors exhibited synergestic effects and may have potential as candidate antiretroviral drugs. Z01 BC 10257-04